Crystal structures of the elusive Rhizobium etlil-asparaginase reveal a peculiar active site

Joanna I Loch,Barbara Imiolczyk,Mariusz Jaskolski,Anna Wantuch,Magdalena Bejger,Joanna Sliwiak,Miroslaw Gilski

doi:10.1038/s41467-021-27105-x

Abstract

Rhizobium etli, a nitrogen-fixing bacterial symbiont of legume plants, encodes an essential l-asparaginase (ReAV) with no sequence homology to known enzymes with this activity. High-resolution crystal structures of ReAV show indeed a structurally distinct, dimeric enzyme, with some resemblance to glutaminases and β-lactamases. However, ReAV has no glutaminase or lactamase activity, and at pH 9 its allosteric asparaginase activity is relatively high, with Km for l-Asn at 4.2 mM and kcat of 438 s−1. The active site of ReAV, deduced from structural comparisons and confirmed by mutagenesis experiments, contains a highly specific Zn2+ binding site without a catalytic role. The extensive active site includes residues with unusual chemical properties. There are two Ser-Lys tandems, all connected through a network of H-bonds to the Zn center, and three tightly bound water molecules near Ser48, which clearly indicate the catalytic nucleophile.

Highlights

Rhizobium etli, a nitrogen-fixing bacterial symbiont of legume plants, encodes an essential Lasparaginase (ReAV) with no sequence homology to known enzymes with this activity
We report that ReAV is an allosteric asparaginase with high pH optimum and with Km of 4.2 mM at pH 9, and that it is a metalloprotein with a puzzling active site
The plant supplies the bacteria with carbon sources and L-Glu, while R. etli supplies ammonia, L-Asp and L-Ala[37]

Summary

Introduction

A nitrogen-fixing bacterial symbiont of legume plants, encodes an essential Lasparaginase (ReAV) with no sequence homology to known enzymes with this activity. Highresolution crystal structures of ReAV show a structurally distinct, dimeric enzyme, with some resemblance to glutaminases and β-lactamases. ReAV has no glutaminase or lactamase activity, and at pH 9 its allosteric asparaginase activity is relatively high, with Km for L-Asn at 4.2 mM and kcat of 438 s−1. The serendipitous detection of strong antileukemic properties of guinea pig serum[2] was attributed by Broom et al.[3] to L-asparaginase activity, and low (μM) Km bacterial asparaginases (Class 1 in revised classification4) of type II5, notably E. coli EcAII, but not type I (e.g. EcAI with mM Km6) became potent drugs in the treatment of acute lymphoblastic leukemia (ALL)[7]. It was realized that plants possess different, Ntn-type, asparaginases (some of which, e.g. in legumes, are K+ dependent8,9) but close homologs of plant-type (or Class 2) asparaginases were discovered in bacteria, e.g. EcAIII10,11

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Conclusion